Figure 6 Caspase-3 activation as determined by flow cytometry. Top four panels: flow cytometric analyses of procaspase-3. Sarcomatoid and 3-Methyladenine clinical trial epithelioid VX-661 clinical trial cells showed a similar baseline expression. In both cell types, a

subpopulation lost expression after selenite treatment. Gray histograms show the negative controls for the immunostaining. Bottom four panels: flow cytometric analyses of caspase-3 activation. Selenite treatment caused the appearance of a distinctly positive subpopulation in the epithelioid cells, whereas the sarcomatoid cells showed a small positive subpopulation that was not distinctly separated from the main peak. Three independent experiments were performed. All eight panels are derived from the same experiment. Divergent data have been published regarding the role of caspases in selenite-induced apoptosis. Several studies have shown that selenite causes a caspase-independent apoptotic cell death [6, 18, 40], whereas others have shown caspase-dependence [9, 17, 36, 57]. We report that caspase-3 was activated in a sub-population of epithelioid cells, but little reactivity was seen in sarcomatoid cells. The limited caspase activation in sarcomatoid cells was surprising. A possible explanation could be an upregulation of Inhibitor of Apoptosis (IAP) family members such as survivin and XIAP. Earlier studies

have found that overexpression of IAP family members is common in mesothelioma cells [58–61]. Inhibition of cathepsin Staurosporine supplier B but not of cathepsins D and E caused increased loss of δΦm Cathepsins are a group of proteases that are physiologically present in lysosomes, and may be released upon stimuli such as oxidative stress [62]. Cells that were pretreated

with cathepsin B inhibitor CA-074 Me showed slightly less apoptosis after selenite exposure (Figure 1). In the sarcomatoid cells, this was reflected in correspondingly increased viability. In the epithelioid cells, the viable proportion decreased slightly instead. Interestingly, when selenite mafosfamide was combined with the cathepsin B inhibitor, the loss of δΦm was greater than with any other inhibitor (Table 2). Cathepsin D and E inhibitor Pepstatin A did not affect the induction of apoptosis by selenite, nor did it alter the loss of δΦm. Signs of autophagy were not detected Autophagy is a form of programmed cell death in which cells do not exhibit apoptotic characteristics. Kim et al have shown that selenite induces autophagy in glioma cells [38]. We wanted to investigate whether some of the cell death that we observe could be due to autophagy. Cells were stained with monodansyl cadaverine and analysed with confocal microscopy for the appearance of granules that might represent autophagic vesicles.